Chromatographic assay systems employed as rapid assay devices are one of several means for detecting the presence of a given analyte in a biological sample. One advantage to these systems is that the execution of these assays does not use additional specialized equipment or trained personnel. Another advantage is the great variety of analytes that can be detected using this type of assay. The use of rapid chromatographic techniques for detection of the presence of an analyte in a biological sample has thus progressed beyond the bounds of the clinical laboratory, as assay devices employing these techniques have been found to be especially valuable in “point of care” situations such as the doctor's office or home settings.
The typical rapid chromatographic tests utilize either a “sandwich” assay or a “competition” assay to detect the presence of a desired analyte. In the sandwich assay, an analyte is bound, or “sandwiched,” between an unlabeled first binding partner and a labeled second binding partner. For example, an analyte, such as an antibody to HIV, can be captured by a first binding partner, in this case, an HIV antigen immobilized on a membrane. The antibody-antigen complex can then be detected by a second binding partner having a label, such as another HIV antigen tagged with a colored particle.
In contrast, during the competition assay, the analyte in the sample competes with a labeled analyte, or labeled analogue to the analyte, for a binding partner immobilized on a solid support. A greater concentration of analyte in the sample results in a lower signal in the assay, as the labeled analytes are competed away from the binding partner on the solid support (i.e., the signal produced during a competition assay decreases as the concentration of analyte in the sample increases). Thus, the sandwich assay provides a qualitative assessment with great sensitivity, while the competition assay provides a quantitative measure of analyte concentration.
Regardless of the analyte-detecting method used, the rapid assay devices currently available are often categorized into one of three basic formats: the “dipstick” format, the “flow through” format, and the “lateral flow” format. The “dipstick” format (exemplified in U.S. Pat. Nos. 5,275,785, 5,504,013, 5,602,040, 5,622,871 and 5,656,503) typically consists of a strip of porous material having a sample receiving end, a reagent zone and a reaction zone. The sample is wicked along the assay device starting at the sample-receiving end and moving into the reagent zone. The analyte to be detected binds to a reagent incorporated into the reagent zone, preferably a labeled binding partner, to form a complex. Typically, these binding pairs are antibody:antigen complexes, or a receptor:ligand complexes having a label such as a colloidal metal incorporated into the reagent portion of the complex. The labeled binding partner-antigen complex then migrates into the reaction zone, where the complex is captured by another specific binding partner firmly immobilized in the reaction zone. Retention of the labeled complex within the reaction zone thus results in a visible readout.
The “flow through” format (U.S. Pat. No. 4,020,046) also utilizes porous solid phase materials. This assay format usually has a porous membrane that contains an immobilized binding partner positioned above an absorptive layer. Once the sample has been added to the membrane surface, the analyte of interest reacts with the immobilized binding partner to form an analyte-binding partner complex. The complex is visualized by addition of a second binding partner having a label, such as an enzyme, one or more dye particles or various colloidal metals. The absorptive layer acts as a sink for excess assay reagents, and can be used to regulate the flow rate of the reactants to achieve optimal reaction between the analyte and the binding partner. In this format, the sensitivity of the readout can be improved by “washing” the membrane with additional solution to reduce any nonspecific binding of the label, or to remove any other materials which can interfere with the assay readout.
The “lateral flow” format (see U.S. Pat. Nos. 5,075,078, 5,096,837, 5,354,692 and 5,229,073) utilizes a porous solid phase material and has a linear construction similar to that of the dipstick assay format: a sample application site, a reagent releasing site and a reaction site. However, instead of vertically wicking the samples up the “dipstick,” the lateral flow format allows a sample to flow laterally across the porous solid phase material. The sample is applied directly to the application site and the analyte of interest flows laterally to the reagent-releasing site, and forms a complex with a labeled binding partner. The analyte:binding partner complex then migrates into the reaction site where it is captured by a second, immobilized binding partner and detected.
The conventional rapid assays are a popular choice for determining the presence of a given analyte in samples provided at the “point of care” sites because they are relatively easy to use, do not use specialized equipment or personnel, and produce results in a short amount of time. For example, simple and rapid immunoassay devices for infectious diseases such as AIDS have been available for almost a decade. However, the existing rapid tests are not without their shortcomings. Most importantly, the sensitivity of such devices has often been questioned, due to various limitations with the currently available formats (Giles et al. (1999) Journal of Medical Virology 59:104-109). In addition, there are several practical limitations to the use of these assay devices inherent in the design of the assay format, as exemplified below.
The dipstick format, which was originally designed for urine analysis, uses a relatively large volume of sample for analysis. This is a considerable limitation to use of such a device for analysis of serum or blood samples. In contrast, assay devices based on the flow-through format reduce the volume requirement of samples significantly. However, the flow-through format cannot be employed in a truly self-contained device. In devices based on the flow-through format, the detecting reagent (i.e. the labeled binding partner) is not directly incorporated into the porous solid matrix of device and thus must be provided separately. This leads to additional limitations regarding reagent stability, if the detecting reagents are provided in liquid form, or issues surrounding the proper preparation and handling of the detecting reagent, if provided in a dried form.
The lateral flow format overcomes both the sample volume problem of the dipstick format, as well as the detecting reagent issue of the flow-through format. However, the lateral-flow format does not allow for a washing step, as inherent in the flow-through format. Any interfering species, such as particulate or colored material introduced by the sample solution, or unbound label, can potentially interfere with the readout of the assay device. As a result, the lateral flow format often employs filtration during the assay procedure, e.g., using specially coated filters to remove potential interfering species prior to detection of the analyte. (see, for example, U.S. Pat. Nos. 4,933,092, 5,452,716, and 5,665,238).
A number of clinical conditions are (or could be) monitored using one or more rapid assay devices. For example, Helicobacter pylori has been identified as a pathogen leading to chronic gastritis, peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma (Huang et al. (1998) Gasteroenterology 114: 1169-79). The conventional “gold standard” tests typically involve invasive endoscopy, followed by histology, culture or rapid urease tests, all of which necessitate a hospital laboratory setting and specially trained medical personnel. On the other hand, the near-patient whole blood or serum/plasma based rapid test devices that have recently become available have not lived up to expectations. There are mixed reports regarding the performances of these kits, particularly in correlation to the ethnic profile of sera being examined. Although some of these rapid test kits perform with approximately 90% sensitivity, often these same kits are compromised by lower performances in specificities, especially when used in different geographical territories. The reverse is also true of kits with high specificities but low sensitivities (see Enroth et al. (1997) J. Clin. Micro. 35: 2695-97; Stone et al. (1997) Eur. J. of Gastroenterol. & Hepatology 9: 257-260; Hackelsberger et al. (1998) Helicobacter 3: 179-183; Leung et al. (1998) J. Clin. Micro. 36:3441-3442). For example, when used to test Asian populations, kits developed using Western serum panels were found to have poorer performance profiles ranging from 63-84% in sensitivity and 82-84% in specificity, considerably lower than those recorded for Western serum panels (Leung, supra). There is an obvious need for an accurate rapid test device for global use, that is both sensitive and specific without compromising one feature for the other.
Tuberculosis (TB) is another example of a clinical condition that would benefit from an improved rapid assay device (for a review of current diagnostic tests, see Andersen et al. (2000) Lancet 356:1099-1104). The re-emerging of this chronic disease is believed to be due largely to the emergence of drug-resistant strains of M. tuberculosis, in concert with a demonstrated increase in risk of infection among human immunodeficiency virus (HIV)-infected populations (for reviews, see Daley et al. (1992) New Engl. J Med. 23;326(4):231-235; Havlir and Barnes (1999) New Engl. J Med. 4;340(5):367-373; Schaaf et al. (1996) Trop Med. Int. Health Oct; 1(5):718-722; and Selwyn et al. (1989) New Engl. J Med. 2;320(9):545-550). Acid-fast bacilli (AFB) microscopy employing a Ziehl-Neelson staining protocol is currently the primary diagnostic and monitoring technique, despite the inherent lack of sensitivity and stringent assay requirements (Perkins (2000) Int. J. Tuberc. Lung Dis. 4(12):51-57; Periera et al (2000) J. Clin. Microbiol. 38:2278-2283). On the other hand, the usefulness of currently available serological tests is debated (Freeman et al (1999) J. Clin. Microbiol. 37(6):2111-2112; Rasolofo and Chanteau (1999) J. Clin. Microbiol. 37(12):4201; Desem and Jones (1998) Clin. Diag. Lab. Immunol. 5:531-536). A recent evaluation of seven currently available serological tests revealed that the sensitivities of such tests are lower than previously reported (Pottumarthy et al. (2000) J. Clin. Microbiol. 38(6):2227-31). Furthermore, the sensitivities of standard serological tests are often heavily diminished in tuberculosis patients co-infected with HIV. There is still an unmet need for new rapid assay devices, particularly those that provide a rapid, inexpensive and accurate test for the diagnosis of TB.